Bilayer Order Research Articles

Coordination of bilayer properties by an inward-rectifier K+ channel is a cooperative process driven by protein-lipid interaction

Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K+ channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K+ flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however, unknown. To investigate, we generated KirBac1.1 samples with different concentrations of 13C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered trans-gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.

The Influence of Lipid Electric Charge on the Binding of Aβ(1-42) Amyloid Peptide to Bilayers in the Liquid-Ordered State.

The amyloidogenic Aβ peptides are widely considered as a pathogenic agent in Alzheimer's disease. Aβ(1-42) would form aggregates of amyloid fibrils on the neuron plasma membranes, thus perturbing neuronal functionality. Conflicting data are available on the influence of bilayer order on Aβ(1-42) binding to membranes. In the present study, a biophysical approach was used in which isothermal calorimetry and surface pressure measurements were applied to explore the interaction of Aβ(1-42) in either monomeric, oligomeric, or fibrillar form with model membranes (bilayers or monolayers) in the liquid-ordered state that were either electrically neutral or negatively charged. In the latter case, this contained phosphatidic acid, cardiolipin, or ganglioside. The calorimetric studies showed that Aβ(1-42) fibrils, oligomers, and monomers could bind and/or be inserted into bilayers, irrespective of electric charge, in the liquid-ordered state, except that monomers could not interact with electrically neutral bilayers. The monolayer studies in the Langmuir balance demonstrated that Aβ(1-42) aggregation hindered peptide insertion into the monolayer, hindered insertion in the decreasing order of monomer > oligomer > fibril, and that lipid composition did not cause large differences in insertion, apart from a slight facilitation of monomer and oligomer insertion by gangliosides.

Effect of the Molecular Weight of Polyelectrolyte and Surfactant Chain Length on the Solid-Phase Molecular Self-Assembly.

Solid-phase molecular self-assembly (SPMSA) is emerging as an efficient approach, leading to scale-span self-assembled supramolecular films. With SPMSA, freestanding macroscopic supramolecular films can be formed upon mechanically pressing the precipitates formed with polyelectrolytes and oppositely charged surfactants. Herein, we report that the film formation ability and the mechanical strength of the resultant film depend highly on the surfactant chain lengths and the molecular weight of polyelectrolytes. A coarse-grained molecular dynamics study revealed that the longer surfactant chains are beneficial for the ordered assembly of surfactant bilayers in the film, whereas the larger molecular weight of PE favors the enhanced mechanical strength of the film by promoting the long-range order of the surfactant bilayers. The current results disclosed the physical insight of the surfactant chain length and the molecular weight of polyelectrolytes into the film structure and mechanical strength, which is of practical importance in guiding the creation of SPMSA materials.

The role of lipid oxidation on electrical properties of planar lipid bilayers and its importance for understanding electroporation

Electroporation is a useful tool for the manipulation with the cell membrane permeability. Underlying physicochemical processes taking place at the molecular level during electroporation are relatively well studied. However, various processes remain unknown, one of them is lipid oxidation, a chain reaction that causes degradation of lipids, and might explain the long-lasting membrane permeability after the electric field has ceased. The aim of our study was to observe the differences in the electrical properties of planar lipid bilayers, as in vitro cell membrane models, due to lipid oxidation. Phospholipids were chemically oxidized and oxidation products were analysed using mass spectrometry. Electrical properties, resistance R (Ω) and capacitance C (F) were measured using an LCR meter. Using a previously developed measuring device, a linear increasing signal was applied to a stable bilayer in order to measure its breakdown voltage Ubr (V) and lifetime tbr (µs). We observed an increase in conductance and capacitance of the oxidized planar lipid bilayers when compared to their non-oxidized counterparts. With increasing lipid oxidation, the core of the bilayer becomes more polar, and consequently more permeable. Our findings can explain the long-lasting permeability of the cell membrane after electroporation.

Excitonic topological order in imbalanced electron-hole bilayers.

Correlation and frustration play essential roles in physics, giving rise to novel quantum phases1-6. A typical frustrated system is correlated bosons on moat bands, which could host topological orders with long-range quantum entanglement4. However, the realization of moat-band physics is still challenging. Here, we explore moat-band phenomena in shallowly inverted InAs/GaSb quantum wells, where we observe an unconventional time-reversal-symmetry breaking excitonic ground state under imbalanced electron and hole densities. We find that a large bulk gap exists, encompassing a broad range of density imbalances at zero magnetic field (B), accompanied by edge channels that resemble helical transport. Under an increasing perpendicular B, the bulk gap persists, and an anomalous plateau of Hall signals appears, which demonstrates an evolution from helical-like to chiral-like edge transport with a Hall conductance approximately equal to e2/h at 35 tesla,where e is the elementarycharge and h is Planck's constant. Theoretically, we show that strong frustration from density imbalance leads to a moat band for excitons, resulting in a time-reversal-symmetry breaking excitonic topological order, which explains all our experimental observations. Our work opens up a new direction for research on topological and correlated bosonic systems in solid states beyond the framework of symmetry-protected topological phases, including but not limited to the bosonic fractional quantum Hall effect.

Interdependence of cholesterol distribution and conformational order in lipid bilayers.

We show, via molecular simulations, that not only does cholesterol induce a lipid order, but the lipid order also enhances cholesterol localization within the lipid leaflets. Therefore, there is a strong interdependence between these two phenomena. In the ordered phase, cholesterol molecules are predominantly present in the bilayer leaflets and orient themselves parallel to the bilayer normal. In the disordered phase, cholesterol molecules are mainly present near the center of the bilayer at the midplane region and are oriented orthogonal to the bilayer normal. At the melting temperature of the lipid bilayers, cholesterol concentration in the leaflets and the bilayer midplane is equal. This result suggests that the localization of cholesterol in the lipid bilayers is mainly dictated by the degree of ordering of the lipid bilayer. We validate our findings on 18 different lipid bilayer systems, obtained from three different phospholipid bilayers with varying concentrations of cholesterol. To cover a large temperature range in simulations, we employ the Dry Martini force field. We demonstrate that the Dry and the Wet Martini (with polarizable water) force fields produce comparable results.

Moiré-driven multiferroic order in twisted CrCl3, CrBr3 and CrI3 bilayers

Layered van der Waals materials have risen as a powerful platform to engineer artificial competing states of matter. Here we show the emergence of multiferroic order in twisted chromium trihalide bilayers, an order fully driven by the moiré pattern and absent in aligned multilayers. Using a combination of spin models and ab initio calculations, we show that a spin texture is generated in the moiré supercell of the twisted system as a consequence of the competition between stacking-dependent interlayer magnetic exchange and magnetic anisotropy. An electric polarization arises associated with such a non-collinear magnetic state due to the spin–orbit coupling, leading to the emergence of a local ferroelectric order following the moiré. Among the stochiometric trihalides, our results show that twisted CrBr3 bilayers give rise to the strongest multiferroic order. We further show the emergence of a strong magnetoelectric coupling, which allows the electric generation and control of magnetic skyrmions. Our results put forward twisted chromium trihalide bilayers, and in particular CrBr3 bilayers, as a powerful platform to engineer artificial multiferroic materials and electrically tunable topological magnetic textures.

Raman spectroscopic studies on the evolution of interlayer coupling and stacking order in twisted bilayers and polytypes of WSe2

Twisted 2D bilayers of van der Waals materials, a new class of quantum materials, offer pioneering advances in the field of nanoelectronics and photonics. As these layered materials can have various preferential stacking configurations with varying electronic behavior, it is important to have a characterization technique that can unambiguously probe the stacking order and interlayer interactions in 2D materials and twisted 2D homobilayers. In this work, we show that by using Raman spectroscopy, we can probe variations in the interlayer coupling of bilayer WSe2 stacked at different twist angles. The interlayer interactions are weakest at a twist angle of 30°, and the twisted bilayer system is almost equivalent to two decoupled monolayers of WSe2. Also we demonstrate Raman mapping as a quick imaging tool with capabilities of clear distinction between 2H and 3R polytypes of bilayer WSe2 and can be used to study various kirigami structures and bilayer nucleation centers commonly observed during chemical vapor deposition-based growth of WSe2. This work proves to be beneficial in the characterization of twisted bilayers of 2D materials and offer key insights into the optoelectronic properties of 2D materials and heterostructures.

Interactions of polycyclic aromatic hydrocarbons and their nitro derivatives with bilayer and monolayer models of fungal membranes

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous persistent organic pollutants forming as byproducts in combustion processes. These compounds can become nitrated in the environment forming even more toxic nitro derivatives. Both PAHs and nitro-PAHs accumulate in the soils which can lead to the pollution of vast areas of arable land. The best means of their elimination from the environment is bioremediation that is the degradation by soil microorganisms. Important key players with high prospects are non-ligninolytic fungi, which can incorporate PAHs and their derivatives into their plasma membranes, oxidize and metabolize to nontoxic products. The accumulation of PAHs in the membrane can, however, be toxic to the fungal cell, but the mechanisms of the PAH-membrane lipid interactions and how they influence the membrane phase state and properties are still poorly understood. To shed light on these questions we employed fungal plasma membrane models and studied their interactions with selected PAHs and nitro-PAHs. Two complementary model systems were investigated: giant unilamellar vesicles (GUV) and Langmuir monolayers. We find that the native PAHs induce phase separation and the formation of solid ordered phase in the GUVs, whereas the nitro-derivatives do not disturb the liquid crystalline state of the membranes. The Langmuir monolayer studies indicate that some PAHs preferably partition in the liquid-expanded phase while others in the liquid-condensed domains. The possibility of the incorporation of PAHs into the condensed domains implies that these compounds can influence the order in the lipid bilayer and affect the function of the lipid rafts in plasma membranes.

Asymmetric effects of amphipathic molecules on mechanosensitive channels

Mechanosensitive (MS) ion channels are primary transducers of mechanical force into electrical and/or chemical intracellular signals. Many diverse MS channel families have been shown to respond to membrane forces. As a result of this intimate relationship with the membrane and proximal lipids, amphipathic compounds exert significant effects on the gating of MS channels. Here, we performed all-atom molecular dynamics (MD) simulations and employed patch-clamp recording to investigate the effect of two amphipaths, Fluorouracil (5-FU) a chemotherapy agent, and the anaesthetic trifluoroethanol (TFE) on structurally distinct mechanosensitive channels. We show that these amphipaths have a profound effect on the bilayer order parameter as well as transbilayer pressure profile. We used bacterial mechanosensitive channels (MscL/MscS) and a eukaryotic mechanosensitive channel (TREK-1) as force-from-lipids reporters and showed that these amphipaths have differential effects on these channels depending on the amphipaths’ size and shape as well as which leaflet of the bilayer they incorporate into. 5-FU is more asymmetric in shape and size than TFE and does not penetrate as deep within the bilayer as TFE. Thereby, 5-FU has a more profound effect on the bilayer and channel activity than TFE at much lower concentrations. We postulate that asymmetric effects of amphipathic molecules on mechanosensitive membrane proteins through the bilayer represents a general regulatory mechanism for these proteins.

Spectroscopic signatures of bilayer ordering in native biological membranes

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (Lo) and liquid-disordered (Ld) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, Lo microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In 13C-13C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.

Tuning Magnetic Order in CrI3 Bilayers via Moiré Patterns

AbstractCommensurable twisted bilayers can drastically change the magnetic properties of chromium trihalide layered compounds, which opens novel opportunities for tuning magnetic states through layer rotations. Here, a mathematical approach to obtain moiré patterns in twisted hexagonal bilayers by performing a certain commensurable rotation θ over one layer is presented. To test the approach, moiré structures with and 32.20° in the phases R and C2/m of CrI3 are obtained via the related methodology. For comparison purposes, a non‐shifted CrI3 structure is also considered. Electronic and magnetic properties of the so‐obtained systems are computed by ab initio methodologies. Results show the presence of rotation‐angle‐dependent magnetic configurations and steep modifications of the dispersion bands due to variations in the nearest and next nearest distances among layers of Cr atoms. Modifications obtained from these commensurable rotations are discussed on the basis of competition among different energy contributions due to changes in the atomic neighborhood.

Structural determination of model phospholipid membranes by Raman spectroscopy: Laboratory experiment.

In an upper-division interdisciplinary laboratory experiment, students use Raman spectroscopy to highlight how the overall structure and conformational order of lipid bilayers can be influenced by their individual phospholipid composition. Students prepare a supported lipid bilayer, as a model cell membrane, by spreading liposomes made of various phospholipids on a solid support. The characterization of phospholipid bilayers, a major component of cellular membranes, can advance our fundamental understanding of important biological phenomena, with significant implications in various fields including drug delivery and development. We use Raman spectroscopy as an analytical tool to investigate the structural and packing properties of model cell membranes. The spectral frequency, intensity, and line-width of lipid Raman bands are extremely sensitive to structural alterations. This experimental module effectively exposes students to the fundamentals of Raman spectroscopy and teaches students the importance of interdisciplinary education as they integrate concepts from chemical structure, molecular interactions, and analytical spectroscopic techniques to gain a more holistic understanding of biological membrane properties.

Frank-van der Merwe growth in bilayer graphene

Frank-van der Merwe growth in bilayer graphene

Modulation of Phospholipid Bilayer Properties by Simvastatin

Simvastatin (Zocor) is one of the most prescribed drugs for reducing high cholesterol. Although simvastatin is ingested in its inactive lactone form, it is converted to its active dihydroxyheptanoate form by carboxylesterases in the liver. The dihydroxyheptanoate form can also be converted back to its original lactone form. Unfortunately, some of the side effects associated with the intake of simvastatin and other lipophilic statins at higher doses include statin-associated myopathy (SAM) and, in more severe cases, kidney failure. While the cause of SAM is unknown, it is hypothesized that these side effects are dependent on the localization of statins in lipid bilayers and their impact on bilayer properties. In this work, we carry out all-atom molecular dynamics simulations on both the lactone and dihydroxyheptanoate forms of simvastatin (termed "SN" and "SA", respectively) with a pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer and a POPC/cholesterol (30 mol %) binary mixture as membrane models. Additional simulations were carried out with multiple simvastatin molecules to mimic in vitro conditions that produced pleiotropic effects. Both SN and SA spontaneously diffused into the lipid bilayer, and a longer simulation time of 4 μs was needed for the complete incorporation of multiple SAs into the bilayer. By constructing potential mean force and electron density profiles, we find that SN localizes deeper within the hydrophobic interior of the bilayer and that SA has a greater tendency to form hydrogen-bonding interactions with neighboring water molecules and lipid headgroups. For the pure POPC bilayer, both SN and SA increase membrane order, while membrane fluidity increases for the POPC/cholesterol bilayer.

How Photoswitchable Lipids Affect the Order and Dynamics of Lipid Bilayers and Embedded Proteins.

Altering the properties of phospholipid membranes by light is an attractive option for the noninvasive manipulation of membrane proteins and cellular functions. Lipids with an azobenzene group within their acyl chains such as AzoPC are suitable tools for manipulating lipid order and dynamics through a light-induced trans-to-cis isomerization. However, the action of these photoswitchable lipids at the atomic level is still poorly understood. Here, liposomes containing AzoPC, POPE, and POPG have been characterized by solid-state NMR through chemical shift and dipolar CH order parameter measurements. Upon UV-light illumination, an efficient trans-to-cis conversion can be achieved resulting in a localized reduction of the CH order parameter within the bulk lipid acyl chains. This effect is even more pronounced in liposomes containing the integral membrane protein E.coli diacylglycerol kinase. The protein responds to the light-induced trans-to-cis isomerization by a site-specific increase in the molecular dynamics as observed by altered cross peak intensities in NCA spectra. This study represents a proof-of-concept demonstration for the use of photoswitchable lipids to modulate membrane properties by light for inducing dynamic changes within an embedded membrane protein.

Dehydration induced dynamical heterogeneity and ordering mechanism of lipid bilayers.

Understanding the influence of dehydration on the membrane structure is crucial to control membrane functionality related to domain formation and cell fusion under anhydrobiosis conditions. To this end, we perform all-atom molecular dynamic simulations of 1,2-dimyristoyl-sn-glycero-3-phosphocholine dimyristoylphosphatidylcholine lipid membranes at different hydration levels at 308 K. As dehydration increases, the lipid area per head group decreases with an increase in bilayer thickness and lipid order parameters indicating bilayer ordering. Concurrently, translational and rotational dynamics of interfacial water (IW) molecules near membranes slow down. On the onset of bilayer ordering, the IW molecules exhibit prominent features of dynamical heterogeneity evident from non-Gaussian parameters and one-dimensional van Hove correlation functions. At a fully hydrated state, diffusion constants (D) of the IW follow a scaling relation, D∼τα -1, where the α relaxation time (τα) is obtained from self-intermediate scattering functions. However, upon dehydration, the relation breaks and the D of the IW follows a power law behavior as D∼τα -0.57, showing the signature of glass dynamics. τα and hydrogen bond lifetime calculated from intermittent hydrogen bond auto-correlation functions undergo a similar crossover in association with bilayer ordering on dehydration. The bilayer ordering is accompanied with an increase in fraction of caged lipids spanned over the bilayer surface and a decrease in fraction of mobile lipids due to the non-diffusive dynamics. Our analyses reveal that the microscopic mechanism of lipid ordering by dehydration is governed by dynamical heterogeneity. The fundamental understanding from this study can be applied to complex bio-membranes to trap functionally relevant gel-like domains at room temperature.

Mechanisms Underlying Drug-Mediated Regulation of Membrane Protein Function

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. The regulation of membrane protein function thus may involve mechanisms ranging from drug binding to specific sites, over more non-specific drug-protein interactions, to drug-induced alteration of bilayer properties. To tease apart these mechanisms, we examine how the prototypical bacterial potassium channel, KcsA, is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). Comparing the relationships between changes in bilayer properties and different KcsA gating steps, and the drugs effects on the bilayer environment reveal that drug-induced regulation of membrane protein function indeed involves a combination of non-specific but direct and bilayer-mediated mechanisms. Both mechanisms are likely to be important whenever there is overlap between a drug's bilayer-modifying and clinical concentration ranges. Using Isothermal Titration Calorimetry (ITC) we determine the binding affinity of the drugs for purified, detergent-solubilized KcsA, which we compare to the affinity for the lipid bilayer in order to separate these direct and bilayer-mediated mechanisms for regulating KcsA function.

Roles of histidine charge and cardiolipin in membrane disruption by antimicrobial peptides Gaduscidin-1 and Gaduscidin-2

Gad-1 and Gad-2 are helical, histidine-rich antimicrobial peptides (AMPs) from paralogous genes in cod. 15N and 2H solid state nuclear magnetic resonance (NMR) were used to characterize their lipid-bound structures and lipid interactions. Gad-1 was found to position in-plane in POPC: POPG bilayers. Gad-1 displayed greater effects than Gad-2 on lipid acyl chain order of POPE: POPG and POPE: POPG: CL bilayers, in keeping with its greater activity against E. coli. The effect of Gad-1 and Gad-2 on lipid bilayer order was only weakly affected by changes in pH, and hence changes in histidine charge. This was somewhat surprising for Gad-2 as this peptide's biological activity has been shown to be greater at low pH and thus the finding may point to the existence of functional interactions with non-lipid components of bacteria. The incorporation of cardiolipin into POPE: POPG bilayers in such a way as to preserve the overall charge of the bilayers did not alter Gad-1's effects on lipid acyl chain order parameters, which report on motions on the 10−5 s timescale. When cardiolipin and Gad-1 were both present, there were subtle changes on membrane dynamics at other timescales.

Stacking-dependent topological phase in bilayer MBi2Te4(M=Ge,Sn,Pb)

Inspired by the breakthroughs of twisted bilayer graphene, stacking-dependent phenomena are recently emerging as fascinating objects of condensed matter research, harboring a variety of new physics. However, current studies are restricted to electronic, superconducting and magnetic properties. Here, using first-principles calculations, we reveal the connection between stacking order and topological property in bilayer $M\mathrm{B}{\mathrm{i}}_{2}\mathrm{T}{\mathrm{e}}_{4}(M=\mathrm{Ge},\mathrm{Sn},\mathrm{Pb})$. We show the stacking order determines the topological property. Upon changing the stacking order, one can achieve topological phase transition between trivial and nontrivial states. We unveil that this stacking-dependent topological property is attributed to the interlayer $\mathrm{Te}\ensuremath{-}{p}_{z}$ orbitals coupling, which is closely related to the stacking order. Our work not only expands the scope of stacking-dependent properties but also provides a promising experimental platform to study this novel stacking-dependent topological property.